Reductive prodrugs are harmless in their native state but kill both growing and non-growing tumor cells upon reduction. Cancer cure requires selective killing of all cancerous cell with minimal side effects, but this is rarely achieved because most drugs cannot reach all the cells within solid tumors (present in >90% human cancers), and are not selective, being activated also by normal human cells. Thus, a drug/catalyst regime is needed that specifically targets tumors and permits visualization of tumor barriers to drug penetration. This proposal is concerned with a newly discovered nitro-prodrug [6-chloro-9-nitro-5-oxo-5H-benzo[a]phenoxazine (CNOB)] and an evolved high-activity nitroreductase discovered and evolved by the PI. CNOB is an effective drug with the unique quality that its activated product is fluorescent and can be visualized in living animals. Novel methods of drug/catalyst delivery to tumors and removal of tumor barriers are also proposed. Four aims will be pursued. The first two involve in vitro studies: the generality of effectiveness against different cancers, and identification of metabolites. Aim 3 deals with specific delivery of the drug/catalyst to tumors. Hyaluronan-labeled nanoliposomes that target the CD44 receptor over-expressed in nearly all cancers will be used to deliver the drug, the catalyst, or both. However, since some normal cells could also express CD44, harmful non-target delivery could occur. Another approach will therefore also be employed that targets two receptors typically overexpressed in cancer cells. While normal cells may express one of these receptors, they would be highly unlikely to express both. In this approach, CNOB will be delivered by CD44-targeting liposomes, and the catalyst by the cytokine induced killer cells that target the tumor-specific NKG2D receptors. This way the normal cells might receive one or the other component of this therapy (the drug or the catalyst) but not both, thus escaping harm. The pharmacokinetics of the drug will be examined for the delivery methods. Aim 4 will dissect the drug and activated product reach within the tumors and will utilize state of the art microscopic and in vivo imaging technology (confocal, IVIS, intravital, autoradiography of tumor sections) and staining (differential fluorescent labels, staining of blood vessels, regions of hypoxia and necrosis) to detect the tumor regions hindering penetration. Removal of these barriers will be attempted by, for example, use of collagenases to dissolve clots, and restoration of normal vasculature in the tumor. Effective means for treating cancer in mice will result, paving the way for human trials of this exciting new cancer therapy. Relevance to public health. Cancer drugs are often unsatisfactory for two reasons: they also harm the non-cancerous cells;and they fail to kill all the cells within a tumor. The drug under study is harmless to normal cells, and will kill cancer cells because a specially developed enzyme will be delivered specifically only to them. This drug can also be seen inside the body;this will allow detection of penetration barriers within the tumors and their elimination.